In complex eukaryotes, 5'-UTRs can be very long and can harbor short Open Reading Frames (ORFs) designated as upstream ORFs (uORFs) ( Young and Wek, 2016 Wethmar, 2014). Non-canonical initiation is also associated with extended 5' Un Translated Regions (5'-UTRs) on mRNAs ( Sendoel et al., 2017 Young and Wek, 2016). The bulk of eukaryotic mRNAs transitions follow this route, but deviations from the canonical route are common, and normally associated with translation under stress conditions ( Starck et al., 2016 Shatsky et al., 2010). The pathway described above is called the canonical, 5'-end and cap-dependent translation route of initiation ( Hinnebusch, 2014). A full (80S) ribosome primed with mRNA and Met-tRNA i Met at the P-site then transitions to the elongation phase ( Wang et al., 2019 Voorhees and Ramakrishnan, 2013). A second GTP-regulated step, catalyzed by initiation factor eIF5B, is then required for the recruitment of the large (60S) ribosomal subunit ( Pestova et al., 2000 Lee et al., 2002). This conformational change is accompanied by the release of eIF1, eIF2 and GDP, leaving the Met-tRNA i Met at the P-site of the 40S base paired with the AUG codon ( Aitken and Lorsch, 2012). Once the AUG codon is detected, a structural transition in the 48S from an open, scanning-competent conformation to a closed, scanning-arrested conformation occurs ( Hussain et al., 2014). Eukaryotic mRNAs are then docked to the 43S-PIC at their 5' ends, forming the 48S complex ( Hinnebusch, 2017). Initiation is significantly expanded in eukaryotes, with two GTP-regulated steps required for the correct positioning of the first aminoacyl-tRNA responsible for setting up the correct reading frame on the messenger RNA (mRNA) ( Jackson et al., 2010 Aitken and Lorsch, 2012 Myasnikov et al., 2009 Hinnebusch, 2014).Įukaryotic initiation starts when the 40S subunit together with the initiation factors eIF1, eIF1A, eIF3, eIF5 and the Ternary Complex (TC) (eIF2–Met-tRNA i Met–GTP) form the 43S Pre- Initiation Complex (43S-PIC), which is competent for mRNA recruitment ( Jackson et al., 2010). Although complex, translation in eukaryotes conserves four main phases that are also found in its prokaryotic counterparts, namely: initiation, elongation, termination and recycling ( Schmeing and Ramakrishnan, 2009). In eukaryotes, especially in animals, this machinery is complex and sophisticated, involving large, multi-component protein factors that assist in the operation of eukaryotic ribosomes ( Hashem and Frank, 2018). As strict cellular parasites, viruses rely on capturing cellular ribosomes to gain access to the host machinery for protein production ( Jan et al., 2016). This diversity is especially overwhelming in RNA viruses that infect animal hosts ( Shi et al., 2016 Dolja and Koonin, 2018). Metagenomic studies of environmental samples have uncovered a great diversity of viruses that have a pervasive presence in the biosphere ( Zhang et al., 2019 Zhang et al., 2018 Greninger, 2018). The structures and accompanying functional data illustrate the importance of 5'-UTR regions in translation regulation and underline the relevance of the untapped diversity of viral IRESs. The IRES features a novel extended, multi-domain architecture, that circles the 40S head. Using a combination of electron cryo-microscopy (cryo-EM) and reconstituted translation initiation assays with native components, we characterized how a novel IRES at the 5'-UTR of a viral RNA assembles a functional initiation complex via an uAUG intermediate. The use of structured RNA sequences, called Internal Ribosomal Entry Sites (IRES), in viral RNAs is a widespread strategy for the exploitation of eukaryotic initiation. The initiation stage of translation is especially targeted as it involves multiple steps and the engagement of numerous initiation factors. To ensure this control, diverse strategies of cellular mimicry and/or ribosome hijacking have evolved. Taking control of the cellular apparatus for protein production is a requirement for virus progression.
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